CN105085631A - Polypeptide capable of specifically targeting HER2 protein and application of polypeptide - Google Patents

Abstract

The present invention relates to a polypeptide specifically targeting HER2 (human epidermal growth factor receptor 2) protein and its application. The polypeptide is shown in the following general formula: X ₁ X ₂ X ₃ X ₄ X ₅ LX ₆ X ₇ X ₈ NPX ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ ; the present invention also relates to a nucleotide sequence encoding a polypeptide, as well as an expression vector and a host cell for expressing the polypeptide; the present invention also relates to the polypeptide and its bivalent Body or multivalent body and the pharmaceutical composition formed as targeting polypeptide and preparation capable of killing cancer cells and imaging preparation and its application, the polypeptide of the present invention can play a targeting role on HER2 positive tumor cells with strong selectivity , and the peptide involved in the present invention can be prepared by chemical synthesis, and has high purity, small molecular weight, strong specificity, no immunogenicity, and safety and reliability.

Description

A kind of polypeptide specifically targeting HER2 protein and its application

technical field

The present invention relates to the field of medicinal chemistry, in particular to a polypeptide, in particular to a polypeptide specifically targeting HER2 protein and its application.

Background technique

Cancer is a leading cause of death worldwide. Data show that in 2012 there were approximately 14.1 million new cancer cases and 8.2 million cancer deaths worldwide, compared with 12.7 million and 7.6 million in 2008. The most common cancers diagnosed worldwide were lung cancer (1.8 million, 13%), breast cancer (1.7 million, 11.9%), and colorectal cancer (1.4 million, 9.7%), and the leading cause of death was lung cancer (1.6 million, 19.4%), liver cancer (800,000, 9.1%) and gastric cancer (700,000, 8.8%). Based on available data, the International Agency for Research on Cancer predicts that due to global population growth and aging, by 2025, the number of new cancer cases worldwide will be as high as 19.3 million per year. In 2012, more than half of the global total of new cancer cases and cancer deaths occurred in underdeveloped regions, accounting for 56.8% and 64.9%, respectively.

At present, there are three main treatment methods for malignant tumors: surgery, chemotherapy, and radiotherapy, among which chemotherapy is the fastest-growing method in tumor treatment in recent years. However, while chemotherapy drugs kill tumor cells, they also kill normal cells in the human body, which has severe side effects. Therefore, research on targeted anticancer drugs and molecular probes is imperative.

The prerequisite for the success of cancer targeted therapy is to identify the target of treatment. HER2 (human epidermal growth factor receptor 2, also known as ErbB2) protein is a 185kDa transmembrane glycoprotein receptor tyrosine kinase, located in the cell membrane, with an extracellular ligand binding region, a single transmembrane region and an intracellular casein amino acid kinase active region. HER2 is involved in signal transduction pathways, activation of which leads to a cascade of phosphorylated kinases leading to cell growth and differentiation. HER2 is overexpressed in various tumors such as breast cancer, ovarian cancer, gastric cancer and uterus. HER2 has become an important target in the treatment of cancer.

Peptides are easy to synthesize in large quantities, have small molecular weight, strong tissue permeability, and can prevent non-specific uptake by the reticuloendothelial system. In addition, peptides can be chemically modified to change their affinity, charge, hydrophobicity, stability, and solubility, which can be modified for in vivo use through iterative optimization. In tumor-targeted therapy, peptide drugs and diagnostic probes have shown strong advantages and are an attractive alternative to antibodies. In terms of application, the combination of polypeptides and radionuclides can be used as probes for molecular imaging; some naturally occurring polypeptides have been used as delivery agents; peptides of reproductive hormones and their derivatives can be used for tumor targeting therapy. Therefore, the efficient discovery of valuable active peptides has become an important task for cancer diagnosis and treatment.

CN104130315A discloses a polypeptide specifically targeting HER2 (human epidermal growth factor receptor 2) protein, said polypeptide is shown in the following general formula: X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ RX ₈ YWX ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ RX ₁₈ X ₁₉ X ₂₀ X ₂₁ YX ₂₂ , can target HER2 positive cells. Although it discloses that the polypeptide has a targeting effect for treating cancer, its interaction with HER2 is not high enough, and its targeting effect is relatively poor.

Contents of the invention

The purpose of the present invention is to provide a polypeptide, a polypeptide specifically targeting HER2 protein and its application, especially products derived from the peptide and capable of binding to HER2 protein, and the above polypeptide or its derivative products in the preparation of targeting Use in HER2 antitumor drugs or imaging preparations.

To achieve this purpose of the invention, the present invention adopts the following technical solutions:

In the first aspect, the present invention provides a peptide targeting HER2 protein, the general formula of its amino acid sequence is: X ₁ X ₂ X ₃ X ₄ X ₅ LX ₆ X ₇ X ₈ NPX ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃

The amino acid residues represented by single-letter symbols in the above peptide chains are defined as follows: L is leucine, N is asparagine, P is proline, and X _1-13 are variable amino acid residues.

As a preferred technical scheme, X1 is _a charged amino acid residue, preferably lysine or aspartic _acid ; X2 is a polar amino acid residue, preferably lysine, threonine or tyrosine; _X3 is an aromatic or aliphatic amino acid residue, preferably phenylalanine or isoleucine; X ₄ is an aromatic or heterocyclic amino acid residue, preferably tyrosine or proline; X ₅ is aromatic or A heterocyclic amino acid residue, preferably tyrosine or proline _; X6 is an aliphatic amino acid residue, preferably glycine or alanine; _X7 is an aliphatic or aromatic amino acid residue, preferably leucine , tyrosine or tryptophan; X ₈ is an aromatic amino acid residue, preferably tyrosine or tryptophan; X ₉ is a polar amino acid residue, preferably threonine or asparagine; X ₁₀ is Polar amino acid residue, preferably threonine or glutamic acid; X ₁₁ is an aromatic amino acid residue, preferably tyrosine or tryptophan; X ₁₂ is an aromatic or polar amino acid residue, preferably benzene Alanine, arginine or glutamine; X ₁₃ is an aromatic or polar amino acid residue, preferably tyrosine, lysine or glutamine.

In the present invention, the source of the polypeptide is a polypeptide library with a library capacity of 2×10 ⁵ established based on the polypeptide sequence DTCPPLMLYNPTTYQM of the HER2 homologous protein EGFR:

The polypeptide sequence DTCPPMLLYNPTTYQM of EGFR is the key site for the formation of EGFR-EGFR dimers and EGFR-HER2 heterodimers, and combines with the II domain of the extracellular domain of HER2 protein. The polypeptide of the present invention is combined with the II structural domain of the extracellular region of HER2, and the polypeptide can specifically bind human HER2 highly expressed positive breast cancer cells, and the binding rate is above 99%.

As a preferred technical solution, the amino acid sequence of the peptide of the present invention is selected from the amino acid sequence shown in one of SEQ ID NO.1-SEQ ID NO.59.

Amino acid sequence:

SEQ ID NO.1 DTFPYLGWWNPNEYRY SEQ ID NO.2 KTIYYLGYYNPNEYRY SEQ ID NO.3 DYIPYLAYYNPNTYFQ SEQ ID NO.4 KKIPYLGWWNPTTWQQ SEQ ID NO.5 DKFPYLALYNPTTWRQ SEQ ID NO.6 DKFPYLALYNPTTYFY SEQ ID NO.7 DKFYPLGYYNPTEWFQ SEQ ID NO.8 DKFYYLAYWNPNEYRQ SEQ ID NO.9 DTFPPLGWWNPTTWFY SEQ ID NO.10 DKFPPLGLYNPNEYFY SEQ ID NO.11 DTFYYLAWWNPNEWRQ SEQ ID NO.12 DTFYYLGYWNPTTYRQ SEQ ID NO.13 DTIYYLAWWNPTTWFY SEQ ID NO.14 DYFPPLGWYNPTTWFY SEQ ID NO.15 DYFPPLGYYNPNEWQY SEQ ID NO.16 DYFPYLAWWNPNEWQQ SEQ ID NO.17 DYFPYLAYWNPNEWQY SEQ ID NO.18 DYFPYLAYWNPNTWRY SEQ ID NO.19 DYFPYLGLYNPNEWFY SEQ ID NO.20 DYFYYLAWWNPNEYFQ SEQ ID NO.21 DYFYYLAWWNPTTWFQ SEQ ID NO.22 DYFYYLGYWNPNEYFY

SEQ ID NO.23 DYIPPLALWNPNEYFY SEQ ID NO.24 DYIPYLALWNPTTYFQ SEQ ID NO.25 DKFPYLALWNPTTYFQ SEQ ID NO.26 DYIPYLAYYNPTEYFQ SEQ ID NO.27 DYIYYLGLWNPTEYQQ SEQ ID NO.28 DYIYYLGLWNPTTWFY SEQ ID NO.29 DYIYYLGYWNPTEWRK SEQ ID NO.30 DYIYYLGYYNPTTYQQ SEQ ID NO.31 KKFPPLALYNPNTWFQ SEQ ID NO.32 KKFPPLGYYNPTEYRY SEQ ID NO.33 KKFPYLAYWNPTTYRY SEQ ID NO.34 KKFPYLGYYNPTEYRQ SEQ ID NO.35 KKFYPLGWWNPNEWRY SEQ ID NO.36 KKFYYLGYWNPNEWQY SEQ ID NO.37 KKFYYLGYWNPNEWRQ SEQ ID NO.38 KKFYYLGYWNPNTWFQ SEQ ID NO.39 KKIPPLAYYNPNTWFY SEQ ID NO.40 KKIPPLGWWNPNTWRY SEQ ID NO.41 KKIPYLAWWNPTTYRQ SEQ ID NO.42 KKIPYLAYYNPNTWQQ SEQ ID NO.43 DKFPYLALYNPTEWQQ SEQ ID NO.44 KKIYYLALWNPTEYRY

SEQ ID NO.45 KKIYYLAWWNPTEYFQ SEQ ID NO.46 KKIYYLGYYNPNEWRQ SEQ ID NO.47 KKIYYLGYYNPTEWRQ SEQ ID NO.48 KTFYYLGWWNPNEWRQ SEQ ID NO.49 KTFYYLGYWNPNEWQY SEQ ID NO.50 KTIPYLAWWNPNTWFQ SEQ ID NO.51 DKFPPLGYWNPTTWQQ SEQ ID NO.52 KYFPPLGWWNPNTYQY SEQ ID NO.53 KYFPPLGYWNPNTWQY SEQ ID NO.54 KYFPYLGWWNPTTWQY SEQ ID NO.55 KYFYYLAYWNPTTYRQ SEQ ID NO.56 KYFYYLGLWNPNEWFQ SEQ ID NO.57 KYIPPLAWWNPTEYQY SEQ ID NO.58 KYIPPLGWWNPTEYRY SEQ ID NO.59 KYIYYLALYNPNTWRY

In the second aspect, the present invention also provides a DNA fragment comprising the amino acid sequence encoding the peptide of the above general formula of the present invention.

As a preferred technical solution, the DNA fragment comprises an amino acid sequence encoding the peptide of the present invention as shown in one of SEQ ID NO.1-SEQ ID NO.59.

In the third aspect, the present invention also provides an expression vector, comprising at least one copy of the DNA fragment encoding the peptide represented by the general formula in the second aspect of the present invention.

As a preferred technical solution, the expression vector of the present invention includes at least one copy of the DNA fragment according to the second aspect of the present invention whose coding amino acid sequence is the peptide shown in SEQ ID NO.1-SEQ ID NO.59.

In the fourth aspect, the present invention also provides a prokaryotic or eukaryotic host cell containing the expression vector as described in the third aspect of the present invention.

In the fifth aspect, the present invention also provides a bivalent body or multivalent body assembled from the peptide described in Formula 1 and the peptide described in SEQ ID NO.1-SEQ ID NO.59 in the first aspect of the present invention.

The above-mentioned bivalent or multivalent body has the characteristic of targeting HER2 positive tumor cells and has lethality to tumor cells.

As a preferred technical solution, the bivalent or multivalent body of the present invention is formed by covalent connection of linking molecules and/or polymers or by mixing with linking molecules and/or polymers and non-covalent linking.

Preferably, the multimer is polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamide-amine dendrimer (PAMAM), polylactic acid (PLA) or polylactic acid- Any one or a mixture of at least two ethanolamines (PLGA).

In the sixth aspect, the present invention further provides a pharmaceutical composition, comprising the amino acid sequence of the first aspect of the present invention being the peptide described in the general formula, the peptide described in one of SEQ ID NO.1-SEQ ID NO.59, or the peptide The bivalent or multivalent body is used as a targeting polypeptide and an agent capable of killing cancer cells.

As a preferred technical solution, the peptide, bivalent body or multivalent body of the present invention is used as a targeting polypeptide, and is conjugated or mixed with an agent capable of killing cancer cells.

Preferably, the preparation is any one of chemical drugs, biopharmaceuticals, nano-medicines, radiopharmaceuticals, photothermal therapy or photodynamic therapy drugs, or carriers encapsulating these drugs that can kill cancer cells;

Further preferably, the preparation is any one of alkylating agents, anti-metabolite drugs, anti-tumor natural drugs, anti-tumor antibiotics, hormones and metal complexes or tumor radiation targeting markers.

Further preferably, the carrier is any one of nanomaterials, liposomes or oily compounds, or a mixture of multiple oily compounds.

The present invention uses the general formula such as the peptide described in one of SEQIDNO.1-SEQIDNO.59, and the bivalent body or multivalent body of the peptide and polymer materials such as nanomaterials and liposomes are conjugated. The present invention involves Peptides, bivalents or multivalents can make the compound generated after conjugation more stable transported to target cells in vivo. The peptides, bivalents or multivalents involved in the present invention can also be mixed with oily compounds or mixtures of multiple oily compounds, and the peptides involved in the present invention can also make the resulting mixture transported to the target more stably in the body cell.

In the seventh aspect, the present invention further provides another pharmaceutical composition, which comprises the amino acid sequence of the first aspect of the present invention is the peptide described in the general formula, the peptide described in SEQ ID NO.1-SEQ ID NO.59 or a bivalent or multivalent form of said peptide; and an imaging agent.

Preferably, the peptide, bivalent or multivalent is conjugated or mixed with an imaging agent.

Preferably, the imaging agent is any one of radionuclide, radionuclide label or molecular imaging agent.

In the eighth aspect, the present invention also provides that the amino acid sequence of the first aspect of the present invention is the peptide described in the general formula, the peptide described in SEQ ID NO.1-SEQ ID NO.59, or the bivalent or multivalent body of the peptide used in the preparation Use in medicaments or imaging preparations for the treatment, prevention or diagnosis of cancer.

As a preferred technical solution, the cancer described in the present invention is a cancer with overexpression of HER2.

Preferably, the cancer is breast cancer, lung cancer, gastric cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, leukemia, bladder cancer or cervical cancer.

The peptide of the present invention has the function of targeting HER2 protein, and can be used as a target to increase the content of drugs or drug-loaded carriers such as nanomaterials, liposomes, etc. in HER2-positive cells, and then add pharmaceutically acceptable excipients or adjuvants to make new and more effective targeted anticancer drugs.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The polypeptide of the present invention can target HER2 positive cells and has strong selectivity, and the polypeptide of the present invention can be prepared by chemical synthesis, has high purity, small molecular weight, strong specificity, and no immunogenicity, Safe and reliable;

(2) The polypeptide of the present invention can bind to human HER2-positive breast cancer cells, with a binding rate of over 99%, and has little binding effect with 293A cells, a normal human embryonic kidney cell with low expression of HER2, indicating that the peptide of the present invention can specifically target HER2 The polypeptide has a selective binding effect on HER2;

(3) The polypeptide of the present invention has the property of targeting HER2-positive tumor cells, and also has the property of killing tumor cells. As a targeting polypeptide, it can be used for targeted therapy of tumors or molecular imaging and diagnosis of targeted tumors.

Description of drawings

Fig. 1 (A) is the action signal of the control group 0.01mMPBS to human HER2 positive breast cancer cells (SKBR3 cell line) detected by flow cytometry; Fig. 1 (B) is the detection of fluorescein isothiocyanate by flow cytometry The binding effect of (FITC)-labeled anti-HER2 antibody on human HER2-positive breast cancer cells (SKBR3 cell line); The binding effect on human HER2 positive breast cancer cells (SKBR3 cell line); Fig. 1 (D) is the detection by flow cytometry of SEQ ID NO.2 labeled with fluorescein isothiocyanate (FITC) on human HER2 positive breast cancer cells ( SKBR3 cell line); Fig. 1 (E) is the binding effect of SEQ ID NO.3 labeled with fluorescein isothiocyanate (FITC) detected by flow cytometry on human HER2-positive breast cancer cells (SKBR3 cell line); Fig. 1 (F) is the binding effect of SEQ ID NO.4 labeled with fluorescein isothiocyanate (FITC) to human HER2-positive breast cancer cells (SKBR3 cell line) detected by flow cytometry; Fig. 1 (G) is the direct binding rate square diagram.

Fig. 2 (A) is the signal of the action of 0.01mMPBS of the control group on human HER2-negative embryonic kidney normal cells (293A cell line) detected by flow cytometry; Fig. 2 (B) is the detection of FITC-labeled anti-HER2 by flow cytometry The binding effect of the antibody on human HER2-negative embryonic kidney normal cells (293A cell line); Figure 2 (C) is the detection of FITC-labeled SEQ ID NO. Binding effect; Figure 2 (D) is the binding effect of FITC-labeled SEQIDNO.2 on human HER2-negative embryonic kidney normal cells (293A cell line) detected by flow cytometry; Figure 2 (E) FITC is detected by flow cytometry Labeled is the binding effect of SEQIDNO.3 on human HER2-negative embryonic kidney normal cells (293A cell line); Figure 2 (F) is the detection of FITC-labeled SEQIDNO.4 on human HER2-negative embryonic kidney normal cells ( 293A cell line); Figure 2(G) is a histogram of the binding rate.

Fig. 3 is the binding effect of FITC-labeled SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 detected by immunofluorescence method on human HER2-positive breast cancer cells (SKBR3 cell line);

Among them, the nuclei were stained with hoechst reagent; A-D are cells treated with SEQ ID NO.1 polypeptide and hoechst, where A is the binding site of SEQ ID NO.1 polypeptide, which mainly exists in the cell membrane, B is the nucleus, C is the whole cell in bright field, D It is the overlay of A-C; E-H is the cells treated with SEQ ID NO.2 polypeptide and hoechst, where E is the binding site of SEQ ID NO.2 polypeptide, which mainly exists in the cell membrane, F is the nucleus, G is the whole cell in bright field, and H is the Overlay of E-G; I-L is the cells treated with SEQ ID NO.3 polypeptide and hoechst, where I is the binding site of SEQ ID NO.3 polypeptide, which mainly exists in the cell membrane, J is the nucleus, K is the whole cell in bright field, L is I-K Superimposed diagram; M-P is the cell treated with SEQ ID NO.4 polypeptide and hoechst, where M is the binding site of SEQ ID NO.4 polypeptide, which mainly exists in the cell membrane, N is the nucleus, O is the whole cell in bright field, and P is M-O Overlay graph.

Fig. 4 is the binding effect of FITC-labeled SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 detected by immunofluorescence method on human HER2-negative embryonic kidney normal cells (293A cell line);

Among them, the nuclei were stained with hoechst reagent; A-D are cells treated with SEQ ID NO.1 polypeptide and hoechst, where A is the binding site of SEQ ID NO.1 polypeptide, B is the nucleus, C is the whole cell in bright field, and D is the superposition of A-C Figures; E-H are cells treated with SEQIDNO.2 polypeptide and hoechst, where E is the binding site of SEQIDNO.2 polypeptide, F is the nucleus, G is the whole cell in bright field, H is the overlay of E-G; I-L is SEQIDNO. 3 Cells treated with polypeptide and hoechst, where I is the binding site of the polypeptide of SEQ ID NO.3, J is the nucleus, K is the whole cell in bright field, L is the overlay of I-K; M-P is the polypeptide of SEQ ID NO.4 and hoechst treated , where M is the binding site of the polypeptide of SEQ ID NO.4, N is the nucleus, O is the whole cell in bright field, and P is the overlay of M-O.

Fig. 5 (A) is that surface plasmon resonance (SPR) method detects the binding effect of SEQIDNO.1 to the human HER2 protein of different concentrations; Fig. 5 (B) is that surface plasmon resonance (SPR) method detects that SEQIDNO.2 is to the people of different concentrations HER2 protein binding; Figure 5 (C) is the surface plasmon resonance (SPR) method to detect the binding of SEQIDNO.3 to different concentrations of human HER2 protein; Figure 5 (D) is the surface plasmon resonance (SPR) method to detect SEQIDNO. 4 Binding effect on different concentrations of human HER2 protein.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the embodiments are only intended to illustrate the present invention, rather than to limit the scope of the present invention. The scope of the invention is specifically defined by the appended claims.

Embodiment 1 The synthesis of polypeptide of the present invention

1) Experimental instruments and materials

Dimethylformamide (DMF), piperidine, resin, dichloromethane (DCM), ninhydrin reagent (ninhydrin, vitamin C, phenol), tetramethyluronium hexafluorophosphate (HBTU), six Hydropyridine (piperidine), triisopropylsilane (TIS), ethanedithiol (EDT), anhydrous ether, trifluoroacetic acid (TFA), N-methylmorpholine (NMM), methanol, various amino acids , Peptide solid-phase synthesis tube.

2) Solution preparation

Deprotection solution - hexahydropyridine: DMF = 1:4

Reaction solution——NMM:DMF=1:24

Lysis solution - TFA (92.5%), TIS (2.5%), EDT (2.5%), H ₂ O (2.5%)

Ninhydrin Test Solution—Ninhydrin: Vitamin C: Phenol = 1:1:1

3) Experimental steps

Weigh the resin and put it into the peptide solid-phase synthesis tube (hereinafter referred to as the reactor), and add an appropriate amount of DMF to swell for more than half an hour. DMF was removed, Fmoc deprotection reaction was carried out with deprotection solution, and placed on a shaker for 10 minutes. Take out the protective solution, wash with DMF and DCM for 3 times, take a small amount of resin (about 5-10 mg) from the reactor in a test tube, wash with ethanol for 2 times, detect and record the color with ninhydrin method, prepare for feeding, enter Amino acid condensation reaction. According to the amino acid sequence of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 peptides, take the corresponding amino acids and HBTU (amino acid: HBTU=1:1), dissolve them in the reaction solution, put them into the reactor, and stir the reaction . After 1-2 hours, take a small amount of resin from the reactor into a test tube, wash it twice with ethanol, and detect it with the ninhydrin method. The liquid in the reactor was sucked out, washed twice with DMF and DCM each, to obtain the peptide resin after condensation of the first amino acid. Repeat the above "Fmoc deprotection-amino acid condensation" reaction step on the obtained peptide resin until the last amino acid is reacted to obtain peptides with sequence numbers of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4. After the reaction is complete, wash the resin 2-3 times with DMF and DCM, wash twice with methanol, and continue to drain for 15-20 minutes. The partially synthesized peptide resin was taken out from the reactor, and lysed in the lysate (the lysate was ice-bathed for 20 minutes first) at room temperature for two hours. After the resin was filtered, it was evaporated to dryness in a rotary evaporator, and washed 3 times with anhydrous ether (ice bath). Crude peptides were purified using preparative reverse phase HPLC with >90% purity using HPLC. The resulting pure peptides were identified using mass spectrometry (MS, electrospray).

After the last peptide was synthesized, a part of the peptide resin was taken out and fluorescently labeled with fluorescein isothiocyanate (FITC). First link Fmoc-e-Acp-OH to the polypeptide according to the amino acid coupling method, then take an appropriate amount of HBTU and FITC and dissolve it in the fluorescent coupling solvent, and test the ninhydrin solution after 3-5 hours. After successful labeling, the same method as above was used for cleavage, purification and identification.

The relevant physical and chemical properties of the peptides of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 are shown in Table 1 and Table 2.

Table 1

Table 2

Experimental Example 1 Flow cytometry detection of the binding effects of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 on human HER2-positive breast cancer cells respectively

1. Experimental method

Collect human breast cancer HER2 high expression cell line SKBR3, suspend in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum, the cell density is about 1x10 ⁶ /mL, and divide into four 1.5mL EP tubes, 200μL /Tube. Add fluorescein isothiocyanate (FITC) labeled SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 and anti-HER2 antibody (ebioscience) respectively, the final concentration is 50μM/L, the control group uses the same amount of polypeptide 0.01mMPBS (phosphate buffer pH 7.4) was used instead of the polypeptide. After incubating in an ice bath in the dark for 30 minutes, centrifuge at 1000g for 4 minutes to collect cells, add 1mL PBS to wash, repeat washing 3 times, add 500μL PBS, mix well, and use flow cytometry to detect fluorescence intensity and binding ratio.

2. Experimental results

As can be seen from Figure 1, the peptides of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 can bind to human breast cancer cells with high expression of HER2, and the binding rates are all above 99%, indicating that the peptides of the present invention are used alone on HER2-positive tumor cells have an affinity effect and can be used as a polypeptide targeting HER2.

Experimental Example 2 Flow cytometry method to detect the binding effects of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 on human HER2-negative embryonic kidney normal cells respectively

1. Experimental method

The human embryonic kidney HER2 low-expression cell line 293A was collected, suspended in H-DMEM medium containing 10% heat-inactivated fetal bovine serum, and the cell density was about 1x10 ⁶ /mL, and distributed in four 1.5mLEP tubes. 200 μL/tube. Add fluorescein isothiocyanate (FITC)-labeled SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, anti-HER2 antibody (ebioscience) respectively, the final concentration is 50 μMol/L, the control group uses the same amount of polypeptide 0.01mMPBS (phosphate buffer pH 7.4) was used instead of the polypeptide. After incubating in an ice bath in the dark for 30 minutes, centrifuge at 1000g for 4 minutes to collect cells, add 1mL PBS to wash, repeat washing 3 times, add 500μL PBS, mix well, and use flow cytometry to detect fluorescence intensity and binding ratio.

2. Experimental results

It can be seen from Figure 2 that the peptides of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 have little binding effect on HER2 low-expression human embryonic kidney normal cells 293A cells, indicating that the peptides of the present invention are selective for HER2 The binding effect can be used as a polypeptide specifically targeting HER2.

Experimental Example 3 Immunofluorescence method to detect the binding effects of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 on human HER2-positive breast cancer cells respectively

1. Experimental method

The human breast cancer HER2 high-expressing cell line SKBR3 was suspended in RPMI1640 culture medium containing 10% heat-inactivated fetal bovine serum, and seeded in three confocol small dishes at a density of 3000-5000 cells/dish. After cultivating for 24 hours, aspirate the medium in the small dish, and then add the culture medium containing 50 μM mol/L of fluorescein isothiocyanate (FITC) labeled SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 polypeptide respectively The base was 200 μL, and the control group used the medium containing PBS (phosphate buffer pH 7.4) equal to the amount of the polypeptide, and the nuclei were stained with the hoechst reagent, which was diluted 1:200. Incubate in an ice bath protected from light for 30 minutes. After washing with PBS and repeating the washing 3 times, 200 μL PBS was added, and the fluorescent signal was observed using a confocal laser microscope.

2. Experimental results

It can be seen from Figure 3 that the peptides of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 can act on the surface of human HER2-positive breast cancer cells, indicating that the peptides of the present invention have a binding effect on HER2-positive tumor cells when used alone , can be used as a polypeptide targeting HER2.

Experimental Example 4 Immunofluorescence method to detect the binding effects of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 on human HER2-negative embryonic kidney normal cells respectively

1. Experimental method

The HER2-negative human embryonic kidney normal cell line 293A was suspended in H-DMEM medium containing 10% heat-inactivated fetal bovine serum, and inoculated in three confocol small dishes at a density of 3000-5000 cells/dish. After cultivating for 24 hours, aspirate the medium in the small dish, and then add the culture medium containing 50 μM mol/L of fluorescein isothiocyanate (FITC) labeled SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 polypeptide respectively The base was 200 μL, and the control group used the medium containing PBS (phosphate buffer pH 7.4) equal to the amount of the polypeptide, and the nuclei were stained with the hoechst reagent, which was diluted 1:200. Incubate in an ice bath protected from light for 30 minutes. After washing with PBS and repeating the washing 3 times, 200 μL PBS was added, and the fluorescent signal was observed using a confocal laser microscope.

2. Experimental results

It can be seen from Figure 4 that the peptides of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 do not bind to normal human HER2-negative embryonic kidney cells, indicating that the peptides of the present invention selectively and specifically bind to HER2-positive tumor cells Yes, it can be used as a polypeptide specifically targeting HER2.

Experimental example 5 surface plasmon resonance (SPR) method detects the affinity of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 to human HER2 protein respectively

1. Experimental method

Spot 1mg/mL of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4 peptides and 0.01mMPBS onto the chip, incubate overnight at 4°C under humid conditions, then wash with 0.1mMPBS for ten minutes and 0.01mMPBS for ten minutes. Minutes, finally wash with deionized water for ten minutes, and repeat once, immerse in 0.01mMPBS containing 5% milk, incubate overnight at 4°C, then wash with 0.1mMPBS for ten minutes, 0.01mMPBS for ten minutes, and finally wash with deionized Rinse with water for ten minutes and repeat once, blow dry with nitrogen, and put on the machine. The mobile phase was sequentially passed through 1.25 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL and 20 μg/mL human HER2 protein, and the SPR signal was recorded and analyzed.

2. Experimental results

It can be seen from Figure 5 that the SPR signals of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, and SEQIDNO.4 peptides gradually increase with the increase of human HER2 protein concentration, and SEQIDNO.1 and SEQIDNO.2 are calculated according to the SPR results. , SEQIDNO.3 and SEQIDNO.4 polypeptides have dissociation constant K _D values for HER2 protein: 1.86×10 ^-8 M/L, 8.12×10 ^-8 M/L, 9.06×10 ^-8 M/L, 2.67 ×10 ^-7 M/L, indicating that the polypeptide of the present invention has a strong binding effect on HER2 and can be used as a polypeptide targeting HER2.

The results of Experimental Examples 1-5 show that at the cellular level, both experimental tests of flow cytometry and immunofluorescence techniques show that the polypeptides of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 are specific and HER2 positive Breast cancer cells bind, and bind to the cell membrane surface with HER2 protein expression, but there is no obvious combination with HER2-negative embryonic kidney normal cells; at the molecular level, surface plasmon resonance (SPR) experiments also confirmed that SEQ ID NO.1, SEQ ID NO .2. The polypeptides of SEQ ID NO.3 and SEQ ID NO.4 all have a strong binding effect on HER2 protein, and with the increase of HER2 protein concentration, the binding signal is stronger.

It can be concluded from Experimental Examples 1-5 that the polypeptide of the present invention has the property of targeting HER2-positive tumor cells, so in practical applications, the peptide of the present invention can be used as a targeting polypeptide and combined with a preparation capable of killing cancer cells It can be combined or mixed for targeted therapy of tumors, or can be conjugated or mixed with imaging agents for molecular imaging and diagnosis of targeted tumors.

The applicant declares that the present invention illustrates the process method of the present invention through the above examples, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. a peptide for special target HER2 albumen, is characterized in that, the general formula of described polypeptide is:

X ₁X ₂X ₃X ₄X ₅LX ₆X ₇X ₈NPX ₉X ₁₀X ₁₁X ₁₂X ₁₃

Wherein, L is leucine, and N is l-asparagine, and P is proline(Pro), X _1-13for variable amino-acid residue;

Preferably, X ₁be charge residue residue, be preferably Methionin or aspartic acid; X ₂be polar amino acid residues, be preferably Methionin, Threonine or tyrosine; X ₃be aromatic series or aliphatic amino acid residue, be preferably phenylalanine or Isoleucine; X ₄be aromaticity or heterocyclic amino acid residue, be preferably tyrosine or proline(Pro); X ₅be aromaticity or heterocyclic amino acid residue, be preferably tyrosine or proline(Pro); X ₆be aliphatic amino acid residue, be preferably glycine or L-Ala; X ₇be aliphatics or aromatic amino acid residue, be preferably leucine, tyrosine or tryptophane; X ₈be aromatic amino acid residue, be preferably tyrosine or tryptophane; X ₉be polar amino acid residues, be preferably Threonine or l-asparagine; X ₁₀be polar amino acid residues, be preferably Threonine or L-glutamic acid; X ₁₁be aromatic amino acid residue, be preferably tyrosine or tryptophane; X ₁₂be aromaticity or polar amino acid residues, be preferably phenylalanine, arginine or glutamine; X ₁₃aromaticity or polar amino acid residues, be preferably tyrosine, Methionin or glutamine.

2. peptide according to claim 1, is characterized in that, the aminoacid sequence of described peptide is selected from the aminoacid sequence shown in one of SEQIDNO.1-SEQIDNO.59.

3. a DNA fragmentation, is characterized in that, it comprises the nucleotide sequence of peptide described in any one of coding claim 1-2.

4. an expression vector, is characterized in that, described expression vector contains the DNA fragmentation as claimed in claim 3 of at least one copy.

5. a host cell, is characterized in that, described host cell contains expression vector according to claim 4.

6. the bivalent that the peptide according to any one of claim 1-2 is formed or multivalent, it is characterized in that, described bivalent or multivalent have the characteristic of target HER2 positive tumor cell and have lethality to tumour cell;

Preferably, described bivalent or multivalent are by connecting molecule and/or the covalently bound formation of polymkeric substance;

Preferably, described bivalent or multivalent be by be connected molecule and/or mixed with polymers, non-covalent linking formed;

Preferably, described polymer is the mixing of any one or at least two kinds in polyoxyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamidoamine dendrimer (PAMAM), poly(lactic acid) (PLA) or poly(lactic acid)-thanomin (PLGA).

7. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises the peptide according to any one of claim 1-2 and can kill and wound the preparation of cancer cells;

Preferably, described pharmaceutical composition comprises bivalent according to claim 6 or multivalent and can kill and wound the preparation of cancer cells;

Preferably, described peptide, bivalent or multivalent, as target polypeptide, are puted together mutually with the preparation that can kill and wound cancer cells or mix;

Preferably, described preparation is any one that can kill and wound the chemicals of cancer cells, bio-pharmaceutical, Nano medication, radiopharmaceuticals, photo-thermal therapy or optical dynamic therapy medicine or wrap up in the carrier of these medicines;

Further preferably, described preparation is any one in alkylating agent, antimetabolite, antitumor natural drug, antitumor antibiotics, hormone, metal complex or tumour radiotherapy target marker;

Further preferably, described carrier is nano material, any one in liposome or oiliness compound, or the mixture be made up of multiple oiliness compound.

8. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises peptide described in any one of claim 1-2 and video picture preparation;

Preferably, described pharmaceutical composition comprises bivalent according to claim 6 or multivalent and video picture preparation;

Preferably, described peptide, bivalent or multivalent are puted together mutually with video picture preparation or are mixed;

Preferably, described video picture preparation is any one in radionuclide, radioisotope labeling thing or molecular image preparation.

9. any one of claim 1-2 or peptide according to claim 6, bivalent or multivalent for the preparation of the purposes in treatment, prevention or the medicine of diagnosing cancer or video picture preparation.

10. purposes according to claim 9, is characterized in that, described cancer is the cancer of HER2 process LAN;

Preferably, described cancer is any one in mammary cancer, lung cancer, cancer of the stomach, liver cancer, colon and rectum carcinoma, the esophageal carcinoma, leukemia, bladder cancer or cervical cancer or nasopharyngeal carcinoma.